Type 1 diabetes (T1D) is a T cell mediated autoimmune disease, where insulin-producing pancreatic beta-ceils are destroyed by autoaggressive CD4 and CD8 lymphocytes. Importantly, autoreactive T cells are found in the peripheral blood of most individuals, but are usually not activated. Thus, true tolerance in the sense of complete absence of autoreactive lymphocytes by thymic negative selection or peripheral deletion appears to be uncommon for certain autoantigens. Several issues are still unclear and will be addressed by this proposal. One can assume that any autoaggressive response will initially target only a few autoantigens before it engulfs and spreads to other antigens released during the inflammatory process. It will be of high therapeutic and pathogenetic interest, whether initiating lymphocytes directed to the primary autoantigens must be present as "drivers" throughout the pre-diabetic phase. Conversely, we do not know whether antigenic spreading, although it occurs in T1D, is a pathogenetically vital process. One would like to understand if all or how many of the antigenic specificities, (including 'new entries') present in an islet infiltrate or pancreatic draining lymph node contribute to pathogenesis. Our first question is whether an initiating autoantigen needs to be expressed throughout the autoimmune process in order to maintain activated, autoaggressive driver T cells (Aim 1). Second, we would like to ask, how the addition of a non-tolerant autoantigen into an already established autoimmune process will influence (accelerate?) the disease course, since a sufficient number of 'driver clones' seeing other autoantigens has already been established (Aim 2). These studies will use a tetracycline-dependent promoter system to 'subtract' the initiating autoantigen from the autoimmune process at different times using the RIP-LCMV model for type I diabetes (Aim 1) and to express a non-tolerant autoantigen at different phases during spontaneous autoimmunity in the NOD mouse and in this way quantitatively address the pathogenetic importance of antigenic spreading during different phases of the disease process (Aim 2). We believe that these two goals will greatly improve our understanding of how autoantigens are driving an autoaggressive process and during which phases of the diabetogenic response the initiating antigen(s) are important.